All plants require nitrogen (N) as an essential nutrient and are able to acquire N from nitrate (NO3−) and ammonium (NH4−) in the soil. Nitrate acquisition begins with its transport into root cells, accomplished by NO3− transporters. Soil NO3− concentrations can vary by five orders of magnitude and plants have evolved both high-affinity (HATS) and low-affinity (LATS) nitrogen transport systems. These systems are encoded by two distinct gene families: the phylogenetically distinct NRT1(PTR) and NRT2 families. Members of these families also participate in movement of NO3− throughout the plant and within plant cells. Proteins in the CLC transporter family also transport NO3−; these transporters are associated with cytosol to organelle NO3− movement.
The nitrogen transporter “NRT1(PTR)” is a large family of transporters, comprising 53 members in Arabidopsis, 84 members in rice, with NRT1(PTR) members known in several other species. In addition to transporting NO3− coupled to Fr movement, members of the NRT1(PTR) family have been found be a peptide transporter (PTR) capable of transporting di- or tripeptides, amino acids, dicarboxylic acids, auxin, and/or abscisic acid. One aspect of the current invention utilizes transgenic plants constitutively expressing a NRT1(PTR) proteins to increase the biomass of plants and/or to decreasing the time needed to grow plants from seed until plants are mature enough to reproduce (flowering).
The plant biomass grown for commercial use is derived from a broad range of crops. Although a majority of crops around the world are grown for food resources, one use for the non-food portions a plant biomass is a renewable source of energy. This is typically conducted by converting the plant biomass into a biofuel. Well managed biomass systems continually grow/replace biomaterial. Biomass energy systems use different conversion and processing technologies to produce solid, liquid and gaseous biofuels. The plant from which the biomass is derived impacts which processes can be used to covert it to fuel, and also the efficiency of the resulting fuel. Furthermore, different crops and processes will yield different types and volumes of biofuels.
Because biomass can be used to create biofuels, methods of developing plants with increased biomass are of great interest to agricultural researchers and corporations. Moreover, some genetically modified plants that are expressing recombinant proteins have been shown to produce higher yield of biomass. While the number of crops genetically modified to have increased resistance to herbicide, insects, and drought conditions has grown significantly over the past decades, there has not been a similar increase in plants developed specifically to increase biomass.
In the US, renewable energy contributes around 7% to the national energy consumption, and nearly half of this renewable energy is derived from biomass. Currently, forests provide around 129 m dry tons of biomass annually, while agriculture provides a further 176 m dry tons. However, according to a study by the US Department of Agriculture and the US Department of Energy (DOE) these two sources could provide up to one billion dry tons (around 940,000 dry tons) of feedstock each year. On this basis biomass could supply 15% of US energy consumption by 2030. The US has a goal of generating one-third of all its liquid fuel from renewable resources by the year 2025, which could require up to 1 billion tons of biomass annually.
The Medicago truncatula (Medicago) MtNIP gene encodes a protein found in plants that is essential for symbiotic nitrogen-fixing root nodule and lateral root development (Veereshlingam et al., 2004; Yendrek et al., 2010). Plants that have defects in the MtNIP gene are able to initiate, but are unable to complete the development of, symbiotic nitrogen-fixing root nodules. These plants also have lateral roots that are incompletely formed (Veereshlingam et al., 2004). FIG. 1 shows the phenotypes observed.
Three mutant alleles of the MtNIP gene have been identified: nip-1, latd (nip-2) (Bright et al., 2005) and nip-3 (Teillet et al., 2008). Using these 3 alleles, one of the present inventors led a team of researchers to carry out a positional, map-based approach to clone the MtNIP gene (also called NIP/LATD or LATD/NIP) (Yendrek et al., 2010).
The MtNIP gene encodes a protein in the NRT1(PTR) transporter family (Yendrek et al., 2010), primarily composed of proton-coupled low affinity nitrate and di- and tri-peptide transporters (Tsay et al., 2007) (FIG. 2). The family also includes a dicarboxylate transporter (Jeong et al., 2004). Recently, the dual affinity nitrate transporter AtNRT1.1, also called CHL1 because of its ability to transport the herbicide chlorate, has been show to function as a nitrate sensor (Ho et al., 2009; Wang et al., 2009) and as an auxin transporter (Krouk et al., 2010).
One aspect of the present invention provides a means of increasing the biomass of plants, as compared to wild-type plants, by over-expressing the MtNIP gene in the plants. Although not wanting to be bound by theory, this finding has implications for genetically engineering crop plants to have greater yield, and also has implications for genetically engineering plants that might be used in production of biofuels, as well as increasing yield for food, fiber and industrial applications.
Arabidopsis thaliana, a dicot, was adopted by the scientific community as a plant model, with the underlying assumption that for most physiological, developmental and genetic processes, it would behave like other plants. It is now considered the reference plant (NSF, 2002; Flavell, 2005). Knowledge gained through the use of Arabidopsis would be applicable to all plants, including crop plants, even though many crop plants are monocots. This is based on evolutionary principles that state that beneath the diversity present in plants that there are core processes and genetic mechanisms that are conserved among all plants (Flavell, 2005). Large scale genome sequencing of the much larger genomes in crop species has begun to show that these species have conserved genes with Arabidopsis, although there are frequent gene rearrangements and gene duplications when one compares Arabidopsis to a crop species (Ware and Stein, 2003; Flavell, 2005). There are numerous examples of genes being tested first in Arabidopsis, because Arabidopsis is fast, and subsequent testing in a crop species, leading to improvement of the crop species: (Bhatnagar-Mathur et al., 2008; Manavalan et al., 2009; Valente et al., 2009; Hussain et al., 2011).
Other examples of Arabidopsis genes being used to improve crop plants or Arabidopsis research paving the way for translational changes in crop plants can be found in Zhang et al (2004) (Zhang et al., 2004). The article lists specific examples of specific genes that were tested first in Arabidopsis and have been used to improve the crop plants Brassica napus, tomato, rice, wheat, strawberry, maize and tobacco. More recent examples show that ERF transcription factor genes that are involved in regulating stress responses in Arabidopsis have been transformed into crop plants rice (Gao et al., 2008; Zhang et al., 2010; Zhang et al., 2010), tomato, and tobacco (Zhang and Huang, 2010), forage clovers (Abogadallah et al., 2011), and alfalfa (Jin et al., 2010), with benefits to the recipient crop plant. In these cases, the transferred gene did not always come from Arabidopsis, but the groundwork experiments were done in Arabidopsis. Although not wanting to be bound by theory, the behavior of MtNIP-transformed Arabidopsis plants will predict other plants that have MtNIP-transformed into them, or are MtNIP-transformed.